Grant: Discovery Grants Program - Individual

Nucleic acids are tremendously versatile molecules. Aside from their role in information storage and transfer, DNA and RNA play active roles as functional molecules. They can act as catalysts (ribozymes, DNAzymes), participate in ligand binding (aptamers, riboswitches), and play a role as material for building nanoscale molecular devices (DNA origami). However, functional nucleic acids are typically thought of as a black box. The fine details of how these types of molecules work are often unknown. Their structures, how they interact with other molecules and how they fold into an active molecule are often poorly understood. Research in my laboratory aims to tackle this lack of understanding and learn how functional nucleic acid molecules perform their complex tasks. As a model system for discovering how functional nucleic acids work, our work is focusing on understanding how the cocaine-binding aptamer operates.The structure of the cocaine biding DNA aptamer is composed of a three-way junction containing a tandem AG mismatch. An unusual feature of the cocaine-binding aptamer is its binding promiscuity. Originally selected to bind cocaine, this aptamer binds quinine 50-fold tighter. This aptamer is also interesting in that its binding mechanism depends on the length of one of its stems. When stem 1 is three base pairs or shorter, it undergoes ligand-induced folding. When it contains four or more base pairs, the structure is pre-formed. The research proposed here aims to fully define the thermodynamics of binding and folding of the aptamer as the length of stem 1 is varied. We will study the free, quinine-bound and cocaine-bound forms of the aptamer in order to determine how the free energy barrier to binding changes as the binding mechanism changes. The three-dimensional structure of the free and ligand-bound cocaine-binding aptamer will be determined using a combination of nuclear magnetic resonance (NMR) and small angle X-ray scattering (SAXS) techniques. These structures will provide important insights not only into the structure of the aptamer but in how it interacts with different ligands. We also aim to apply our knowledge of the structure and function of this aptamer to assemble a chain of aptamers in a controlled and designed manner. This work will produce fundamental insights in to molecular recognition as well as practical applications in using aptamers as biosensors. A long-term aim of my research program is to expand these studies to other aptamer-ligand systems in order to formulate general rules for how different functional nucleic acid molecules work.